The incidence of severe obesity (BMI > 40 kg/m2) is increasing disproportionately and associated with elevated health care costs. We have reported that fatty acid oxidation (FAO) is depressed in the skeletal muscle of severely obese patients, which may predispose these individuals to ectopic lipid accumulation. Our preliminary findings suggest that this decrement in FAO is due to a reduction in mitochondrial content which is linked with an inability to appropriately induce mitochondrial biosynthesis (metabolic inflexibility). Together, these data are indicative of a metabolic program with severe obesity which favors lipid deposition; in contrast exercise training increases FAO and rescues metabolic flexibility in severely obese patients. These findings have led to our central hypothesis that the depressed FAO and metabolic inflexibility evident in the skeletal muscle of severely obese individuals is due to a reduction in mitochondrial content which stems, at least in part, from an inability to respond appropriately to conditions that induce mitochondrial biosynthesis, with the notable exception of exercise training. Findings could aid in understanding the underlying biology of this condition and designing effective treatments as well as promoting physical activity as an intervention/prevention for severe obesity. Our hypothesis will be tested by the following aims. Aim 1: To determine if the regulation of mtDNA in skeletal muscle is altered with severe obesity in a manner that depresses FAO. a) Is occupancy of the NRF-1 (nuclear respiratory factor -1) binding site on the TFAM (mitochondrial transcription factor A) promoter reduced with severe obesity? b) Is the reduction in TFAM expression linked with methylation of the TFAM promoter region? c) Can TFAM overexpression ameliorate the metabolic phenotype evident with obesity? d) Is insulin-induced mitochondrial biosynthesis impaired in the muscle of severely obese individuals? e) Are the defects discerned in HSkMC evident in-vivo? Aim 2: To determine if there is a coordinated dysregulation of nuclear genes required for mitochondrial biosynthesis with severe obesity. a) Is there a coordinated reduction in the expression of NRF-1 target genes with severe obesity? b) Is NRF- 1 occupancy on genes linked with mitochondrial biosynthesis reduced with severe obesity? c) Can NRF-1 overexpression rescue the decrements in mitochondrial content, FAO, and metabolic inflexibility evident with severe obesity? Aim 3: To determine if components of exercise training can rescue skeletal muscle from the metabolic phenotype/program evident with severe obesity. a) Does contractile activity increase FAO and metabolic flexibility to a similar extent in HSkMC from lean and severely obese individuals? b) Does contractile activity restore obesity-related decrements in TFAM or NRF-1 target genes? c) Will an increase in energy demand reverse the severely obese phenotype? d) Does exercise training (in-vivo) rescue muscle from the metabolic program evident with severe obesity? PUBLIC HEALTH RELEVANCE: We have previously reported that severely obese (BMI > 40 kg/m2) individuals display a decrement in fatty acid oxidation in skeletal muscle which may lead to positive lipid balance and ectopic lipid accumulation. The broad, long-term objective of the current proposal is to discern the cellular mechanisms responsible for the reduction in fatty acid oxidation in the skeletal muscle of severely obese humans and if exercise training functions as an effective intervention. The relevance of this work is that the incidence of severe obesity is increasing disproportionately and associated with elevated health costs; findings obtained could aid in understanding the underlying biology of this condition and designing treatments as well as promoting physical activity as an effective intervention/prevention for severe obesity.